Ca²⁺ ionophore A23187 and thrombin inhibit the pertussis‐toxin‐induced ADP‐ribosylation of the α‐subunit of the inhibitory guanine‐nucleotide‐binding protein and other proteins in human platelets

Abstract

Inhibitory guanine-nucleotide-binding proteins (Gi proteins) are substrates for pertussis toxin and the decreased pertussis-toxin-dependent ADP ribosylation of Gi proteins upon prior specific hormonal stimulation of cells is thought to reflect the receptor-mediated activation of Gi proteins, leading to their subsequent dissociation into ai and p/y subunits. In the present study, the effect of various platelet stimuli on the subsequent pertussis-toxindependent ADP ribosylation of the a subunit of Gi (Gi,) in saponized platelets and platelet membranes were studied. Stimulation of intact platelets with the Ca'+-ionophore A23187 or thrombin, but not phorbol 12,13-dibutyrate, decreased the subsequent pertussis-toxin-dependent ADP ribosylation of Cia in saponin-permeabilized platelets in a time-dependent and dose-dependent manner. Thrombin was more effective than A23187. Parallel measurements of Ca2 mobilization and pertussis-toxin-dependent ADP ribosylation of Gi, in platelets showed that Ca2 + mobilization could only partly account for the decrease in pertussis-toxin-dependent ADP ribosyhtion in platelets stimulated by thrombin. When the ADP-ribosylation reaction was carried out in platelet membranes. a decrease in ADP ribosylation was still observed after stimulation of platelets with thrombin, but not with A23187. In addition to Cia, two other proteins were found to be ADP ribosylated by pertussis toxin; their ADP ribosylation was also decreased after A23187 and thrombin stimulation of platelets. The results indicate that Ca2+ mobilization can decrease the pertussis-toxin-dependent ADP ribosylation of Gi, in saponized platelets; the decrease of pertussis-toxin-dependent ADP ribosylation of Cia after thrombin stimulation of platelets can only, in part, be explained by Ca2+ mobilization and involves additional mechanisms; the decrease in pertussis-toxindependent ADP ribosylation after A23187 and thrombin stimulation is not confined to Gi, and involves other proteins. We conclude that the decrease in pertussis-toxin-dependent ADP ribosylation of G j in thrombinstimulated platelets might not be solely caused by a specific structural change, such as dissociation of Gi. It is likely that A23187 and thrombin stimulation of platelets generates substances which interfere with the ADPribosylating activity of pertussis toxin.Receptor-mediated activation of the trimeric guaninenucleotide-binding (G) proteins leads to their dissociation into the subunits a and[I]. Various techniques are used to identify the coupling of specific receptors to their respective G proteins [2]. Gi proteins (Gil, G j 2 and Gi3) are substrates for pertussis toxin and Gj x-subunits (Cia) are only ADP ribosylated by pertussis toxin when associated with and y subunits [2-41. Therefore, a decreased pertussis-toxin-dependent A D P ribosylation of Gi proteins upon specific hormonal stimulation of cells or cell homogenates is thought to reflect the receptor-mediated activation and dissociation of Gi proteins. It has been shown that prior exposure of platele...

Oxidant-Mediated Activation of Cytosolic Phospholipase A2in Pulmonary Endothelium: Role of Protein Kinase Cα and a Pertussis Toxin–Sensitive Protein

“…In view of this and to gain an insight into the biochemical mechanisms associated with the activation of PKC under oxidant-triggered condition caused by t-buOOH in bovine pulmonary artery endothelial cells, we tested the hypothesis that t-buOOH-mediated activation of an aprotinin-sensitive protease plays a crucial role in activating PKC and subsequently stimulating cPLA 2 activity in the cell membrane, and AA release from the cells. Because a previous study indicated that pretreatment of pertussis toxin in some cells inhibits stimulation of AA release caused by different stimulants (Clark et al 1986;Sies et al 1991), we also determined whether pertussis toxin pretreatment could prevent t-buOOH-induced cPLA 2 activity and AA release in the cells. Our results suggest that treatment of bovine pulmonary artery endothelial cells with t-buOOH stimulates an aprotinin-sensitive protease activity, which activates PKCα and that subsequently phosphorylates 41-kDa α-subunit of Gi (Giα), leading to stimulation of cPLA 2 activity in the endothelial cell membrane and subsequently AA release from the cells.…”

Proteolytic Activation of Protein Kinase Cα by Peroxynitrite in Stimulating Cytosolic Phospholipase A2 in Pulmonary Endothelium: Involvement of a Pertussis Toxin Sensitive Protein

“…In view of this and to gain an insight into the biochemical mechanisms associated with the activation of cPLA 2 under oxidant-triggered condition caused by ONOO - in bovine pulmonary artery endothelial cells, we tested the hypothesis that ONOO - -mediated activation of an aprotinin-sensitive protease plays a crucial role in activating PKC and subsequently stimulating cPLA 2 activity in the cell membrane and AA release from the cells. Since previous studies indicated that pretreatment of pertussis toxin in some cells inhibits AA release caused by different stimulants ( , ), we also determined whether pertussis toxin pretreatment could prevent ONOO - -induced cPLA 2 activity and AA release in the cells. Our results suggest that treatment of bovine pulmonary artery endothelial cells with the oxidant ONOO - stimulates an aprotinin-sensitive protease activity, which activates PKCα and that subsequently phosphorylates the 41 kDa α-subunit of G i (G i α) leading to stimulation of cPLA 2 activity in the endothelial cell membrane and subsequently AA release from the cells.…”